PROJECT SUMMARY/ABSTRACT Peripheral nerve injury (PNI) is a common and complex clinical challenge. Nerve autografts remain the ?gold standard? for reconstruction of PNI with a large gap, but limited availability of donor nerves and several severe donor site morbidities significantly impede their clinic application. The combinatorial use of tissue engineered nerve guidance conduits (NGCs) with supportive cells and bioactive factors has the potential to be alternatives to nerve autografts. Mesenchymal stem cell (MSC)-based therapy has shown promises in regenerative medicine due to their multipotent, immunomodulatory/anti-inflammatory, and regenerative potentials. The therapeutic effects of MSCs most probably rely on their secretome, particularly the secretory extracellular vesicles (EVs) which play important roles in intercellular communication through transfer of various bioactive molecules e.g. lipids, proteins, and noncoding RNAs. Due to their comparable therapeutic efficacy to parental cells, MSC-EVs have been explored as novel cell-free therapeutics for a spectrum of diseases. However, the potential use of MSC-EVs in peripheral nerve regeneration remains largely unexplored. We have recently shown that human gingiva-derived MSCs (GMSCs) and their released EVs displayed comparable therapeutic effects on regeneration of taste bud/taste sensory nerves of rats and repair/regeneration of crush-injured sciatic nerves of mice possibly by promoting reprogramming of myelinated Schwann cells toward a repair phenotype. Our preliminary study showed that infusion of GMSC-EVs into the customized AxoGuard Nerve Connectors significantly improved their efficacy on regeneration of transected rat sciatic nerves. Through proteomic profiling, we identified a group of protein factors significantly enriched in GMSC-EVs, among which milk fat globule-epidermal growth factor-factor VIII (MFG-E8) is particularly interesting because of its pleiotropic biological functions. Our preliminary data also showed that MFG-E8 activated pSTAT3 and upregulated gene expressions involved in regulating repair phonotypic conversion of Schwann cells. We hypothesize that enriched MFG-E8 contributes a major role in GMSC-EVs mediated therapeutic effects on nerve regeneration by promoting the repair phenotypic conversion of Schwann cells. We proposed two specific aims to test the hypothesis: 1) determine the pleiotropic effects of MFG-E8 enriched in GMSC-EVs on the repair phenotypic conversion of Schwann cells; 2) investigate the critical role of MFG-E8 in GMSC EV-mediated therapeutic effects on regeneration of transected rat sciatic nerves. Our long-term goal is to develop a cell-free/stem cell- based product as a potential alternative to nerve autografts for regeneration of PNI with a large gap. Results from this study will lead us to submit an R01 grant application that will allow us to better understand the mechanisms underlying MSC-EV mediated therapeutic effects on nerve regeneration and to further explore the safety and efficacy of MSC-EVs in nerve regeneration in large animals and early human clinical trials.